Research this past year focused on the molecular characterization of glutamate receptors and their function in the auditory system. Four families of inotropic glutamate receptors and eight metabotropic glutamate receptors have now been identified. To characterize the biochemical and cell biological properties of this complex set of proteins, we have developed selective antibodies to most of the inotropic receptor subunits and to two of the metabotropic receptors. This past year we developed antibodies selective for mGluR2 and 3; we found that these receptors are localized in presynaptic terminals and glia as well as in postsynaptic densities. Most neurons express multiple glutamate receptors and several subunits of any inotropic receptor. Our studies of the cerebellar Purkinje neuron show that several glutamate receptors are co-expressed at its two synaptic populations; the climbing fiber input and the parallel fiber input. Immunoprecipitation analyses suggest that multiple AMPA receptor complexes, which differ in their subunit compositions, are expressed in hippocampal neurons. The biochemical properties of the NMDA NR1 splice variants and NR2 subunits were studied. Analysis of the NMDA receptor complex show a modification in the structure after incorporation into the synaptic membrane, as determined by its detergent solubility properties. In the auditory system, cochlear and vestibular ganglion cells express the metabotropic glutamate receptors mGluR1, 3, 4, 5 and 7 in addition to several inotropic glutamate receptors. Hair cells are immunopositive for mGluR1, mGluR2/3 and mGluR5. In the cochlear nucleus, following an auditory nerve lesion there is a general up-regulation of all splice variants of the NMDA receptor subunit, NR1, in the dorsal cochlear nucleus and a general down-regulation in the ventral cochlear nucleus. We are using a differential display to characterize gene expression in the inner ear. Several promising candidate genes that are selectively expressed in the organ of Corti or are down-regulated by ototoxic drug treatment have been identified.